TRAF6 regulates autophagy and apoptosis of melanoma cells through c‐Jun/ATG16L2 signaling pathway

Abstract Autophagy and apoptosis are essential processes that participate in cell death and maintain cellular homeostasis. Dysregulation of these biological processes results in the development of diseases, including cancers. Therefore, targeting the interaction between apoptosis and autophagy offers a potential strategy for cancer therapy. Melanoma is the most lethal skin cancer. We previously found that tumor necrosis factor receptor‐associated factor 6 (TRAF6) is overexpressed in melanoma and benefits the malignant phenotype of melanoma cells. Additionally, TRAF6 promotes the activation of cancer‐associated fibroblasts in melanoma. However, the role of TRAF6 in autophagy and apoptosis remains unclear. In this study, we found that knockdown of TRAF6 induced both apoptosis and autophagy in melanoma cells. Transcriptomic data and real‐time PCR analysis demonstrated reduced expression of autophagy related 16 like 2 (ATG16L2) in TRAF6‐deficient melanoma cells. ATG16L2 knockdown resulted in increased autophagy and apoptosis. Mechanism studies confirmed that TRAF6 regulated ATG16L2 expression through c‐Jun. Importantly, targeting TRAF6 with cinchonine, a TRAF6 inhibitor, effectively suppressed the growth of melanoma cells by inducing autophagy and apoptosis through the TRAF6/c‐Jun/ATG16L2 signaling pathway. These findings highlight the pivotal role of TRAF6 in regulating autophagy and apoptosis in melanoma, emphasizing its significance as a novel therapeutic target for melanoma treatment.

of China, Grant/Award Number: 2022YFC2504700; Science Found for Creative Research Groups of the National Natural Science Foundation of China, Grant/Award Number: 82221002 TRAF6 in regulating autophagy and apoptosis in melanoma, emphasizing its significance as a novel therapeutic target for melanoma treatment.

K E Y W O R D S
apoptosis, ATG16L2, autophagy, c-Jun, melanoma, TRAF6

INTRODUCTION
Cutaneous melanoma is the most lethal skin cancer, accounting for more than three-quarters of deaths from skin cancers. 1 During the past two decades, the incidence of melanoma has increased by approximately 2% annually. According to statistics from the American Cancer Society, more than 100,000 new cases of melanoma are expected to be diagnosed and over 7 thousand deaths from melanoma are expected to occur in 2023 (https://www. cancer.org/). Novel treatment strategies for melanoma have been developed, for example, targeted therapies and immunotherapies, which notably prolong the overall survival of melanoma patients. 2 However, the response rate variability between individuals and drug resistance are commonly observed and result in treatment failure. Therefore, the development of further approaches remains highly important to provide more options for melanoma therapy.
Autophagy is a cellular process involved in the degradation of cellular components within lysosomes under specific conditions. It plays a role in various biological processes related to cancer. 3 Autophagy has been reported to have a dual role in the development of melanoma. In the early stage, decreased autophagy leads to the loss of protective mechanisms in cells, thereby promoting tumor formation. The restoration of autophagy in advanced melanoma may benefit tumor progression and cause drug resistance, 4 while increased autophagy could also induce apoptosis in tumor cells under certain conditions. 5,6 The interplay between autophagy and apoptosis is a complex system. Some specific molecules, such as BCL2 and p62, may be critical links between the two processes. P62 participates in both the selective autophagic degradation and apoptotic pathways by interacting with cell survivalrelated proteins such as Caspase-8, tumor necrosis factor receptor-associated factor 6 (TRAF6), and extracellular signal-regulated kinase (ERK). 7-10 TRAF6, belonging to the tumor cecrosis factor (TNF) receptor-associated factor family, is an important signaling molecule that plays a critical role in regulating various cellular processes, including the immune response, inflammation, and cell survival. TRAF6 interacts with several downstream signaling pathways, including the c-Jun N-terminal kinase (JNK) and NF-kB pathways, 11 which further regulate the transcription of genes involved in cellular processes. For example, TRAF6 can activate the c-Jun pathway by promoting the phosphorylation and activation of JNK, or interact with c-Jun directly and promote its stabilization by preventing its degradation. 12,13 It has been documented that the c-Jun pathway participates in autophagy by modulating the expression of mammalian target of rapamycin (mTOR), a negative regulator of autophagy, or by activating the transcription factor EB (TFEB), a key regulator of lysosomal biogenesis and autophagy. 14,15 Our previous work suggests that TRAF6 is highly expressed in melanoma and enhances its invasion and metastasis abilities. 16 In addition, TRAF6 induces the activation of fibroblasts to cancer-associated fibroblasts in the tumor microenvironment, therefore promoting the malignant phenotype of melanoma cells. 17 Nevertheless, the precise involvement of TRAF6 in the autophagic and apoptotic processes within melanoma cells remains uncertain.
Our transcriptomic data suggest that knockdown of TRAF6 regulates the autophagy and apoptosis pathways in melanoma cells. Herein, we found that TRAF6 knockdown promoted both apoptosis and autophagy. The expression level of Autophagy Related 16 Like 2 (ATG16L2) was decreased in TRAF6-deficient melanoma cells. ATG16L2, a member of the ATG16 protein family, has been documented to participate in the process of selective autophagy, specifically targeting organelles and proteins in intestinal cells. 18 However, the exact role of ATG16L2 in the autophagy process in the context of cancer is not fully understood. Here, we found that suppressing ATG16L2 expression led to elevated apoptosis and autophagy in melanoma cells. The mechanistic investigation further revealed that TRAF6 regulated the expression of ATG16L2 through c-Jun. We next found that cinchonine, a TRAF6 inhibitor, suppressed melanoma tumor growth by enhancing autophagy and leading to apoptosis through the c-Jun/ATG16L2 pathway. In this research, we present novel findings regarding the function of TRAF6 in apoptosis and autophagy. Specifically, we provide the first evidence demonstrating that TRAF6 is involved in both of these cellular processes. Additionally, we show for the first that ATG16L2 is implicated in autophagy and is regulated by the TRAF6/c-Jun pathway in melanoma cells. These findings indicate that targeting the TRAF6/c-Jun/ATG16L2 axis represents a promising therapeutic target for the treatment of melanoma.

TRAF6 regulates the proliferation of melanoma cells
As we previously demonstrated that TRAF6 was overexpressed in melanoma cells, we expressed short hairpin (sh)-TRAF6 in the human melanoma cell lines SK-Mel-5 and SK-Mel-28. Interestingly, we found that the growth of these melanoma cells was significantly inhibited. Therefore, we detected apoptosis in SK-Mel-5 and SK-Mel-28 cells by Annexin V staining, as illustrated in Figures 1A and B. The apoptosis rate increased fivefold in melanoma cells after knockdown of TRAF6 (apoptosis rate: 60-80%) compared with that in sh-Mock cells (10-20%) ( Figures 1C and D). During cell death, the poly (adenosine diphosphate-ribose) polymerase-1 (PARP-1) enzyme facilitates the repair of damaged deoxyribonucleic acid (DNA) and cleavage of PARP-1 is a hallmark of apoptosis. 19,20 In addition to PARP-1, BCL2 and caspase family members also regulate cell death. 21 Hence, we measured the expression of these apoptosis-related proteins by western blotting, which showed that the levels of cleaved PARP, and cleaved Caspase-9 were significantly increased and the level of BCL2 was greatly decreased in TRAF6-knockdown melanoma cells ( Figure 1E). These results suggested that TRAF6 is involved in the proliferation of melanoma cells.

Knockdown of TRAF6 induces autophagy in melanoma cells
The interplay between apoptosis and autophagy is widely acknowledged. Increased autophagy can result in induction of apoptosis through lysosomal activity. 22 Considering this observation, we detected autophagy after knocking down TRAF6. Interestingly, transmission electron microscopy (TEM) analysis suggested that many autophagosomes and autophagic bodies were formed in sh-TRAF6 melanoma cells, as shown in Figure 2A. LC3 is well recognized as a key protein in autophagic activity 23 ; therefore, we evaluated the formation of LC3 puncta and observed an elevation in the number of autophagosomes in cells where TRAF6 was knocked down ( Figure 2B). Elevated LC3 II and decreased p62, which are indicators of autophagy, 24 were found through western blotting ( Figure 2C). These results indicated that autophagy was activated when TRAF6 was knocked down in melanoma cells.

The influence of TRAF6 on gene expression profiles in melanoma cells
To gain deeper insights into the impact of TRAF6 on melanoma cell proliferation, we performed ribonucleic acid (RNA) sequencing of SK-Mel-5 cells as described previously. 17 We performed Kyoto Encyclopedia of Genes and Genomes pathway analysis to analyze the differentially expressed genes identified in our study. The results suggested that the cell growth and death pathway class was altered in TRAF6-knockdown melanoma cells ( Figure S1). Since autophagy was induced, we examined the expression of autophagy-related genes in the sequencing database. Interestingly, the fragments per kilobase of transcript per million mapped reads (FPKM) values of autophagy-related genes such as ATG16L2, ATG4C, ATG2B, ATG10, ATG14, ATG3, ATG3, and BECN1 [25][26][27] were significantly altered in SK-Mel-5 cells with TRAF6 knockdown (Figures 3A  and S2). RT-PCR analysis further identified the differential expression of these genes ( Figure S3), among which ATG16L2 exhibited the greatest decrease in expression in TRAF6-deficient cells, consistent with the sequencing data ( Figure 3B). Analysis of the RNA sequencing data revealed that TRAF6 exerts a regulatory influence on the expression of genes related to autophagy, with ATG16L2 exhibiting the most significant alteration.

Inhibition of ATG16L2 induces autophagy and apoptosis in melanoma cells
ATG16L2 is an autophagy-related protein, but its role in autophagy has not been clarified. ATG16L1-deficient macrophages were found to exhibit impaired autophagy and promote the secretion of inflammatory cytokines. 28 In contrast, knockdown of ATG16L2 was found to increase autophagy in pancreatic acinar cells. 29 Since we found a significant change in ATG16L2 expression in TRAF6knockdown melanoma cells, further investigation of the role of ATG16L2 was deemed highly important. Therefore, we knocked down ATG16L2 in melanoma cell lines, including SK-Mel-5 and SK-Mel-28 ( Figures 3C and D). First, we measured the protein levels of apoptosis markers in ATG16L2-deficient cells. Interestingly, we observed a significant increase in the expression of proapoptotic proteins including cleaved PARP, cleaved Caspase-9 and Bax, as well as decreased levels of antiapoptotic proteins such as BCL2 ( Figure 3E). Next, we tried to determine the function of ATG16L2 in melanoma cells. The level of autophagy was evaluated by TEM, and numerous phagosomes were observed in ATG16L2-knockdown melanoma cells ( Figure 3F). Consistent with our previous findings, these cells expressed a higher level of LC3 II and a decreased level of p62 ( Figure 3G), indicating the regulatory role of ATG16L2 in autophagy. These results support the notion that inhibition of ATG16L2 plays a role in modulating autophagy and apoptosis in melanoma cells.

Cinchonine induces apoptosis and autophagy in melanoma cells
Here, we found that TRAF6 participates in the survival pathway in melanoma cells through ATG16L2, and our previous studies demonstrated that TRAF6 promotes the malignant phenotype of melanoma cells. 16,17 Considering these findings, targeting TRAF6 could be a promising therapeutic approach for melanoma. Hence, we treated melanoma cells with cinchonine, a compound from the Cinchona alkaloid family that binds to the RING domain of TRAF6 and affects its function. 30 As expected, both SK-Mel-5 and SK-Mel-28 cells exhibited an increased apoptosis rate when treated with cinchonine ( Figures 4A and B). After treatment with 100 μM cinchonine, 29.7 ± 0.76% of SK-Mel-5 and 19.5 ± 0.15% of SK-Mel-28 cells were apoptotic. When the cinchonine concentration was increased to 200 μM, apoptosis was detected in 35.02 ± 2.27% of SK-Mel-5 and 30.2 ± 0.21% of SK-Mel-28 cells ( Figures 4C and D), indicating that cinchonine induces apoptosis in melanoma cells in a dose-dependent manner. Consistent with these results, western blotting showed that the levels of cleaved PARP and cleaved Caspase-9 were elevated, while the level of BCL2 was decreased ( Figure 4E), which further demonstrated that cinchonine induces apoptosis in melanoma cells. Next, TEM was performed in melanoma cells treated with cinchonine. Notably, increased accumulation of autophagic bodies and autophagosomes was observed in cinchonine-treated melanoma cells ( Figure 5A). More autophagosomes were found after treatment with 200 μM cinchonine than after treatment with 100 μM cinchonine. Figure 5B illustrates the effects of cinchonine on the levels of LC3 II and p62, two key markers associated with autophagy. Taken together, these data suggested that treatment with cinchonine induced apoptosis and autophagy in melanoma cells.

Cinchonine inhibits the growth of melanoma cells in vitro and in vivo
To investigate the inhibitory role of cinchonine in cell growth, we treated melanoma cells with different concentrations of cinchonine. The growth of SK-Mel-5 and SK-Mel-28 cells was reduced by approximately 70% after treatment with 50 μM cinchonine and by 50% after treatment with 100 μM cinchonine. When the concentration was increased to 150 μM, the growth was almost completely inhibited ( Figure 5C). Then, we treated xenograft-bearing mice with cinchonine (100 mg/kg) and found that tumor growth was significantly suppressed ( Figures 5D and E). To assess the toxicity of cinchonine, we measured the body weight of mice treated with the compound, as TRAF6 is expressed in various cells ( Figure 5F). The data indicate that there was no significant decrease in the weight of the mice, suggesting that cinchonine has a tolerable side effect. Immunohistochemistry was then performed on tumor slices to investigate the role of cinchonine in the expression of TRAF6, c-Jun, and ATG16L2 in tumor tissue. Our results demonstrated that the expression of TRAF6, ATG16L2, and c-Jun was inhibited in mice treated with cinchonine compared with the control group. These findings are consistent with the in vitro results ( Figure S4). Staining of proliferating cell nuclear antigen (PCNA) suggested the presence of fewer proliferating cells in cinchonine-treated tumors ( Figures 5G and H). Moreover, the expression level of Caspase-9 was elevated in cinchonine-treated tumors ( Figure S4). Taken together, these results indicated that cinchonine inhibited the proliferation and induces apoptosis of melanoma cells.

2.7
Cinchonine suppresses the TRAF6/c-Jun/ATG16L2 pathway To better understand the regulatory role of TRAF6 in ATG16L2 mRNA expression, we constructed the pGL3-ATG16L2-luc plasmid ( Figure 6A upper panel). Then pGL3-ATG16L2-luc was cotransected with the Mock or TRAF6 plasmid into HEK293T cells, and the transfected cells were subsequently subjected to a luciferase assay. Overexpression of TRAF6 significantly increased the luciferase activity of the ATG16L2 promoter ( Figure 6A, lower panel). TRAF6 regulates the p38/c-Jun pathway in diverse diseases, with effects including modulating inflammatory pathways in keratinocytes, 31 promoting cell proliferation and progression in colorectal cancer, 32 and inducing angiogenesis. 33 According to the prediction from PROMO (http://alggen.lsi.upc.es/), the ATG16L2 promoter contains several potential binding sites for c-Jun ( Figure 6B). Hence, we first performed a luciferase reporter assay and found that the luciferase activity of the ATG16L2 promoter was elevated after overexpression of c-Jun ( Figure 6C). Then, we designed 5 pairs of primers for chromatin immunoprecipitation (ChIP) assays to examine the effect of TRAF6 on the binding of c-Jun to the ATG16L2 promoter. The results with Primer-4 indicated that TRAF6 knockdown significantly reduced the occupancy of c-Jun in the ATG16L2 promoter in SK-Mel-28 cells ( Figure 6D), suggesting that TRAF6 regulates ATG16L2 expression at the transcriptional level through c-Jun.
Since cinchonine was reported as a TRAF6 inhibitor and was found to induce apoptosis and autophagy, the inhibitory role of cinchonine in melanoma cells might be associated with the ATG16L2 pathway. Therefore, we first measured the expression of ATG16L2 after treatment with cinchonine. As shown in Figure 6E, treatment with 150 μM of cinchonine notably decreased the level of ATG16L2 in melanoma cells. Next, we evaluated the expression of nuclear c-Jun after cinchonine treatment. Interestingly, cinchonine significantly suppressed the nuclear accumu-lation of c-Jun in both SK-Mel-5 and SK-Mel-28 cells ( Figure 6F). ChIP assays with Primer-4 further confirmed that cinchonine suppressed c-Jun binding to the ATG16L2 promote ( Figure 6G). Taken together, these results indicated that cinchonine inhibited the growth of melanoma cells through the TRAF6/c-Jun/ATG16L2 signaling pathway.

DISCUSSION
Apoptosis and autophagy are different processes of cell death. However, there is no clear boundary between these two processes. It is well documented that apoptosis inhibits the development of cancers by suppressing cell proliferation and metastasis. Apoptosis can be mediated by both the intrinsic pathway after stimulation by stressors such as DNA damage, starvation, and oxidative stress and the extrinsic pathways through the binding of death ligands to death receptors. 34 Apoptosis of misplaced cancer cells is a key step in inhibiting metastasis. Therefore, several small molecules targeting apoptotic pathways and their components, including the p53 signaling pathway, BCL2 family members, and cIAPs, have been investigated for cancer therapy. 35,36 Unlike that of apoptosis, the role of autophagy is controversial. Under physiological conditions, autophagy enables the degradation of proteins and organelles to obtain amino acids and macromolecules necessary for cell survival, therefore protecting cells from nutrient deprivation. 37 During the early stage of cancer, reduced autophagic activity increases the proliferative capacity of cells, hence promoting their malignant transformation. In the advanced stage of cancer, autophagy is restored, benefits the survival of starved cancer cells and leads to drug-resistance. 38 Therefore, the role of autophagy is related to the status of cells. Under some conditions, apoptosis and autophagy are interconnected. For example, the proapoptotic protein TRAIL mediates autophagy during lumen formation. 39 Ceramide, which triggers apoptosis, induces macroautophagy. 40 There are several nodes of crosstalk between apoptosis and autophagy such as beclin-1-BCL2 41 and p53. Increased beclin-1 expression induces BCL2 to trigger apoptosis and enhances the effect of anticancer drugs in cervical cancer. 42 Suppression of BCL2 can upregulate the expression of beclin-1, thus leading to apoptosis in breast cancer cells. 43 P53, a tumor suppressor, is well recognized to induce apoptosis. It plays a complex role in autophagy: cytoplasmic p53 inhibits autophagy, while nuclear p53 activates autophagy. 44 In this study, we uncovered that TRAF6 exerts regulatory control over both apoptosis and autophagy in melanoma cells. Downregulation of TRAF6 notably increased the apoptosis rate of cancer cells. Autophagic activity was elevated in TRAF6-deficient melanoma cells, which contained more autophagosomes. TRAF6 is highly expressed in various cancers. TRAF6 suppresses the mitochondrial translocation of p53, thus inhibiting apoptosis in cancer cells. 45 In osteosarcoma cells, knockdown of TRAF6 was found to increase the apoptosis rate. 46 In addition to its established role in apoptosis, TRAF6 has been implicated in the regulation of autophagy under specific conditions. Perturbation of the interaction between AMBRA1 and TRAF6 disrupts autophagy by modulating the K63-linked ubiquitylation of specific proteins. 47 TRAF6 can also interact with ATG9 thereby inducing autophagy. 48 Although several studies have investigated the role of TRAF6 in different cancers, its regulatory role in the crosstalk between apoptosis and autophagy remains unknown. In addition to its direct regulation of apoptosis-or autophagy-related genes, TRAF6 also plays a crucial role in promoting cell survival by activating downstream signaling pathways. For example, in human lung cancer cells, TRAF6 interacts with and ubiquitinates PI3K, leading to phosphorylation of AKT at T308 and S473, which endows cancer cells with resistance to apoptosis. 49 Furthermore, TRAF6 stabilizes myeloid cell leukimia (MCL)-1 through K63-linked polyubiquitination, which promotes cell growth. 50 Notably, TRAF6-dependent phosphorylation of ERK1/2 induces upregulation of MCL-1, ultimately suppressing apoptosis in liver cancer cells. 51 These studies provide compelling evidence for the pivotal role of TRAF6 in regulating cell growth and death. In this study, we present evidence supporting the role of TRAF6 in the downregulation of ATG16L2 through c-Jun, which ultimately triggers both apoptosis and autophagy in melanoma cells. Moreover, we demonstrate that inhibition of TRAF6 with cinchonine leads to a significant reduction in the growth of melanoma cells both in vitro and in vivo.
Our study has certain limitations. TRAF6 has been previously reported to regulate apoptosis by modulating AP-1 expression. In this study, we demonstrated that TRAF6 modulated the expression of c-Jun/ATG16L2, and that knocking down either TRAF6 or ATG16L2 induced apoptosis in melanoma cells. However, the contribution of the ATG16L2 pathway to TRAF6-induced apoptosis remains unclear. In addition, the specific mechanisms by which ATG16L2 regulates apoptosis require further elucidation.

CONCLUSION
Taken together, our results indicate that the TRAF6/c-Jun/ATG16L2 signaling axis plays an essential role in the crosstalk between apoptosis and autophagy in melanoma. Cinchonine, an inhibitor of TRAF6, suppressed the growth of melanoma cells and exhibited antitumor effects, suggesting that targeting TRAF6 is a promising therapeutic strategy in melanoma.

Antibodies and reagents
The primary antibodies used were as follows: anti-TRAF6

Protein preparation and immunoblotting
Cells were lysed using RIPA lysis buffer (P0013D; Beyotime, Shanghai, China), and the protein concentrations were determined using a BCA Protein Assay Kit (Santa Cruz). Nuclear proteins were extracted by NE-PER Nuclear and Cytoplasmic Extraction Reagents (78835; Thermo Scientific, MA, USA) according to the manufacturer's instructions. Proteins were separated by SDS-PAGE and electroblotted onto polyvinylidene fluoride membranes (Millipore, Billerica, MA). The membranes were then incubated with specific antibodies, and the immunoreactions were detected using a Bio-Rad imaging system (Bio-Rad, USA).

5.4
Electrophoretic mobility-shift assays A total of 3 μg of nuclear protein was utilized for analysis with an AP-1 kit (AP-1 IRDye 700, 829−07925; LI-COR Biosciences, NE, USA) in accordance with the manufacturer's instructions. Immunoreactions were then detected using the Bio-Rad imaging system (Bio-Rad, USA).

5.5
Cell counting kit-8 assay 3000 cells/well were plated in 96-well plates and incubated with indicated time. After incubation, cell counting kit-8 (CCK-8) solution (B34304; Bimake, TX, USA) was added and incubated following the manufacturer's instructions. The absorbance of samples was measured at 450 nm using a spectrophotometer (Beckman, USA). Six replicates of each sample were analyzed. at room temperature in the dark, then 300 μL of 1× binding buffer was added to stop the reaction. Then the samples were analyzed using flow cytometry within 20 min.

Transmission electron microscopy
Cells were harvested using trypsin and subsequently fixed with 2.5% glutaraldehyde (pH 7.4) at 4 • C for 2 h. The samples were then forwarded to Wellbio (Changsha, China) for additional processing, which included embedding, sectioning, staining, and imaging using a JEM1400 transmission electron microscope (JEOL USA, MA, USA).

Lentiviral infection
TRAF6-deficient and ATG16L2-deficient cells were generated as described previously. 17 In brief, sh-Mock, sh-TRAF6, or sh-ATG16L2 plasmids were cotransfected with packaging and envelope plasmids (pSPAX2 and PMD2G) into HEK293T cells. The lentiviral particles were harvested at 48 and 72 h posttransfection and stored at −80 • C. Target cells were subsequently infected with the lentiviral particles overnight in the presence of 10 μg/mL polybrene. Replacing the medium with fresh medium containing 2 μg/mL puromycin on the next day. Further experiments were performed using these cells until all control cells became nonviable, which usually occurred within 36−48 h in the presence of puromycin.

Immunohistochemistry
The preparation of paraffin-embedded tumor slices was performed as described previously. 17 The primary antibody against PCNA (YM6090; ImmunoWay Biotechnology Company, TX, USA) was used in this study. The average rate of positive PCNA staining was obtained by randomly selecting and analyzing five regions of each sample

Immunofluorescence
Control and TRAF6-deficient melanoma cells were transfected with mCherry-GFP-LC3 for visualization of free autophagosomes (both GFP and mCherry fluorescence) and autophagosomes that had fused with the lysosomes (autolyosomes; mCherry fluorescence only). Zeiss LSM 710 was used to capture fluorescence images (Oberkochen, Germany).

Quantitative reverse transcription-PCR analysis
The extraction of total RNA and reverse transcription for RT-PCR were performed as described previously. 17 The PCR primers used in the study are listed in Table  S1. Relative mRNA expression was calculated using the 2 −△△CT value. F I G U R E 6 TRAF6 regulates the gene expression profiles of cell growth and death pathway in melanoma cells. (A) Overexpression of TRAF6 increases ATG16L2 luciferase activity. Schematic diagram of pGL3-ATG16L2-luc plasmid construction (A upper panel). The pGL3-ATG16L2-luc and Renilla luciferase gene were cotransfected with Mock or TRAF6 plasmid into HEK293T cells. After 24 h transfection, Firefly luciferase activity was examined through normalized against Renilla luciferase activity as described in section Materials and Methods (A lower panel). The data from multiple experiments are expressed as the mean ± SD (n = 4). Significant differences were evaluated using Student's t-test, ***p < 0.001. (B) Schematic diagram of ATG16L2 promoter and PROMO predicted several binding sites of c-Jun. (C) ATG16L2 luciferase activity is regulated by c-Jun in melanoma cells. The pGL3-ATG16L2-luc and Renilla luciferase gene were cotransfected with Mock or c-Jun plasmid into HEK293T cells. 24 h later, Firefly luciferase activity and Renilla luciferase activity were detected. The data from multiple experiments are expressed as the mean ± SD (n = 3). Significant differences were evaluated using Student's t-test, **p < 0.01. (D) Knockdown of TRAF6 attenuates c-Jun associated with ATG16L2 promoter. ChIP assay was performed to examine the c-Jun recognition of the ATG16L2 promoter as described in section Materials and Methods. The data from multiple experiments are expressed as the mean ± SD (n = 3). Significant differences were evaluated using Student's t-test, *p < 0.05. The data of Primer 1, 2, 3, and 5 were not shown as it did not work. (E) Cinchonine downregulated the expression of ATG16L2 in melanoma cells. Whole cell lysate of SK-Mel-5 and SK-Mel-28 treated with -, 100, and 150 μM cinchonine were extracted and subjected to immunoblot analysis using antibodies to ATG16L2 as described in section Materials and Methods, GAPDH was used as control. (F) Nuclear c-Jun is decreased after cinchonine treatment in melanoma cells. Nuclear protein was extracted from cinchonine-treated melanoma cells and subjected to immunoblot analysis using antibodies to c-Jun or electrophoretic mobility-shift assays as described in section Materials and Methods. Lamin A/C was used as control. (G) Cinchonine reduces c-Jun associated with ATG16L2 promoter. ChIP assay of cinchonine-treated melanoma cells with Primer 4 was performed as described in section Materials and Methods. The data from multiple experiments are expressed as the mean ± SD (n = 3). Significant differences were evaluated using one-way ANOVA, ***p < 0.001.

5.12
Luciferase reporter gene assays The following vectors were used: the pGL3 luciferase reporter vector (E1751; Promega, Madison, WI, USA); the pENTER vector (P100001; Vigene, USA); and the pRLTK Renilla luciferase control reporter vectors (P100001; Promega). HEK293T cells were transfected with pENTER (Mock), the TRAF6 or c-Jun, and pGL3-ATG16L2-luc (constructed in our laboratory), along with pRLTK. TRAF6-deficient melanoma cells were transfected with the pGL3-ATG16L2-luc and pRLTK plasmids. The next day, firefly and Renilla luciferase activity in the cell lysates was measured using the dual luciferase assay kit (E1910; Promega) following the manufacturer's protocol. The luciferase activity in four replicates per transfection was averaged.

ChIP assay
For the ChIP assay, melanoma cells were transduced with the sh-Mock and sh-TRAF6 lentiviral vectors or treated with varying doses of cinchonine and subjected to the EZ ChIP KIT protocol (17−371RF; Millipore). Soluble lysates were incubated with 5 μL of an anti-c-Jun antibody (9165S; CST) overnight at 4 • C in the presence of protease inhibitors. The ATG16L2 promoter regions were amplified by PCR using the primer pairs described in Table S1.

Xenograft model
Six-week-old female athymic BALB/c mice were procured from the Department of Laboratory Animals, Central South University. SK-Mel-5 cells were harvested and rinsed with PBS buffer, and then suspended in cold serum-free DMEM at a concentration of 10 7 /mL. 100 μL of cells were subcutaneously injected into the right flanks of the nude mice. Tumors were measured three times per week using calipers, and tumor volumes were calculated using the following formula: length × width × height × 0.52. Tumor tissues were excised, fixed with 10% buffered formalin, and embedded in paraffin for further hematoxylin and eosin staining or immunohistochemical analysis.

Statistical analysis methods
The statistical results are expressed as means ± standard deviations (SD) values, and the significance of differences was determined using Student's t-test, one-way ANOVA, or two-way ANOVA. Statistical significance was assumed at p < 0.05.